The initial idea of DNA exponential amplification was derived from Khorana's similar process, which is “repair replication of short synthetic DNAs as catalyzed by DNA polymerases” (Kleppe, J. Molec. Biol., 56:341) in 1971. However lacking of syntheses oligonucleotide, heat-resistant polymerase, and the thermal cycler limits its development at that time. Until 1983, Kary Mullis from genetic synthesis lab of Cetus corporation in US had the inspiration to simulate natural DNA double replication process in vitro and conceived cell free DNA amplification by thermocycling-polymerase chain reaction (PCR). The general principle of PCR is to exponential amplify a fragment DNA of bracket sequence with suitable template, two oligo-nucleotide primers, DNA polymerase, four nucleotide-triphosphates, an appropriate buffer, and cycling of DNA denaturation, renaturation, extension steps. Following available of thermal cycler instrument and development of thermostable polymerase Taq, Cetus' scientists eventually succeeded in the PCR and filed first PCR patents (U.S. Pat. Nos. 4,683,195 & 4,683,202) in 1985, certificated in 1987.
The PCR amplification consists of a series of 30-40 cycles of repeated the denature-annealing-extension three temperature steps. (1) Template DNA denature step: The target template melted by heating reaction to 94° C. for 20-30 seconds and double strands disrupted to single strand that ready for primer binding. (2) Primer annealing step: The reaction temperature is cooling to 54° C.-60° C. for 20-40 seconds allowing the primers which complement with preselected target sequence to bind on the single-stranded template. (3) Extension/elongation step: Then the temperature is increased to 72° C.-80° C., a new DNA chain synthesize by adding dNTPs to paired template from the 3′end of primer that bound on the target strand with DNA polymerase at optimum temperature. The many semi-conservative DNA strands that reverse complement to the original template are synthesized in 5′ to 3′ direction after repeated these denature-annealing-extension procedures and new strands also used as next cycles template. Following “n” cycles completed, an exponential increasing in the total number of target fragments between the primers is finally reached at theoretical abundance of 2n. But if “n” are more than 30 rounds of cycles, the primer dimers will become extremely crucial. The more precise formula Y=(1+X)n is available, where Y represents the copy number of products and X represents amplification efficiency which theoretical value of 100%, the n is number of cycles. On the first a couple of cycles, although 100% efficiency lets the products to double increase, but the amount of products still low and signal of amplification remain baseline. After about more than 10 cycles, both the amount and the signal of products enter exponential amplification or called log-phase which is most sensitive and easy test. As accumulation of products and consumption of PCR reagents such as dNTPs and primers, the reaction slow down to achieve linear stage which is no longer increased exponentially. Last stage, PCR enter the plateau, no more product accumulates due to exhaustion of components.
The PCR methods gradually matured and practical following a kind of Taq thermo-stable DNA polymerase was purified from the thermophilic bacterium, Thermus aquaticus, which naturally lives in hot (50 to 80° C.) environments-such as hot springs in 1985-1988. The several other pfu, Vent, Tth polymerase lately was discovered, which is dramatically improved PCR operation. Therefore the journal Science awarded Taq polymerase its first “Molecule of the Year” in 1989. The PCR with extreme high sensitivity and simplicity had spread all over the world and became most important basic techniques in life science field from 1989. Kary Mullis of Cetus, was awarded the Nobel Prize in Chemistry in 1993.
In the next twenty years, up to dozens of new improvements and methods of PCR had emerged and invented, including reverse transcription PCR (RT-PCR), In situ PCR, Ligase chain reaction (LCR), Labeled PCR (Labeled primers, LP-PCR), Reverse PCR (amplification of the unknown sequence outside two primers), Asymmetric PCR, Touchdown PCR, Nested PCR, Recombinant PCR, Multiplex PCR, Immuno-PCR, Differential display PCR, Strand displacement amplification (SDA), Nucleic acid sequence-based amplification (NASBA), Transcript-based amplification system (TAS), Q-beta replicase catalytic RNA amplification, Rolling circle amplification (RCA), Loop mediate isothermal amplification (LAMP), etc. Especially the varieties of real-time fluorescence PCR, which detected amplification progress in “real time”, realize the quantitative leap versus standard PCR qualitative test at its end, such as the fluorescent dye SYBR Green I real-time PCR and various fluorescent probes TaqMan (hydrolysis probe), FRET hybridized probes, and Molecular beacon probes, as described in overview (Maisa L. Wong and Juan F. Medrano, BioTechniques 39:75-85, July 2005). The nucleic acid amplification PCR technology not only greatly improved the DNA cloning technology also revolutionary advanced the sensitivity and efficiency of nucleic acid quantification. Applications of the PCR have been expanded to many fields of biology. Now PCR technique is not single method, but it is a new subject that includes a series of new academic theory, methodology and application. The detailed review also sees the PCR books (Huang Liuyu, et al., “principle, method and application of PCR technology”, Chemical industry press, 2005). The PCR that is widely applied in molecular cloning, sequencing and gene recombination, protein engineering, such as life science research, and medical care, agriculture and forestry, animal husbandry, environment protection, food safety, and many other testing applications, has become the most important core scientific technology of the 21st century. In recent years, for lower reagent consumption and higher speed heating/cooling rates, the PCR based detective devices are developing to the rapid microfluidic miniature PCR (lab—on a chip) and multiple target detection of high throughput PCR chip (PCR—on a chip), as showed in the paper “SURVEY and SUMMARY”, (Da Xing, 2007, Nucleic Acids Res., Vol. 35, No. 13, p 4223-4237). Since the first report of PCR, more than million papers that involved with PCR are published, and more than three to four digits patents or related designs are filed.
After above overview, because the quantity of products at the plateau of even same PCR reaction is magnified into much different, the endpoint detection of conventional PCR can be only qualitative analysis. And the sensitivity of regular 30 of PCR is still not enough to detect the lower than the thousands copies of specimens. Otherwise the primer-dimer (PD) be fast accumulated with big trouble if reaction is excess 30 cycles. For the PD amplification and products aerosol result in false positive results, which is the reason of the conventional PCR only 30 cycles of reaction. Thus the conventional PCR which product should be check by gel electrophoresis is difficult to apply to clinical diagnosis. The kinetic PCR with fluorescence monitoring of amplification progress in real time, which was first presented by Higuchi in 1992, provides a idea in order to solve these problems of endpoint PCR. The real-time fluorescent PCR (qPCR) quantitative analysis is determined through the product quantity of fluorescence detecting (product binding fluorescence intensity) during cycling-time and the fluo-signal is directly related to the starting numbers of gene copies. The initial copies of target are negatively logarithmic scale to the cycling numbers for Ct value (Cycle threshold) when amplification product quantity increase of fluorescence signal achieved the threshold of logarithmic phase. Since the amount of DNA in the reaction double at each cycle. Thus if the template diluted double, relative one more cycle would be need to amplify to the same products or the identical threshold. The detecting of signal corresponding to the amplification products may employ with DNA binding dye as well as fluorescence label probe. The former is based on non-specific fluorescent dye that intercalate with any double-stranded DNA such as SYBR Green I real-time fluorescent PCR (U.S. Pat. No. 6,569,627); The second is sequence-specific DNA probes with a fluorescent reporter at one end and a quencher of fluorescence at opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence. The labeled probe permits detection only after hybridization of the probe with its complementary DNA target. The probe with 5′ fluorescent group, which is inhibited by 3′quencher, can be activated by degradation, such as the Taq enzyme hydrolysis of fluorescent labeled probe PCR was developed by PE company of US (Livak K J, et al., 1995, Genome Res, 7-362) who filed the hydrolysis (trade name: TaqMan) PCR invention in 1997 (U.S. Pat. No. 6,485,903). Following Epoch company improved the binding efficiency of MGB probe PCR invention (U.S. Pat. No. 7,205,105). The quenched label probe can also be activated by structure change, such as the a stem loop structure of hybridization probe-Molecular beacon (Tyagi S, et al., 1996, Nat Biotechnol comes 3-308), which's 5′ end sequence bound and inhibited by 3′quencher sequence, and was applied for Molecular beacon invention in 1999 (U.S. Ser. No. 05/925,517). The other series of the double probe hybridization (FRET), Scorpion probe, Sunrise-Primer, the fluorescent Lux-Pimer, etc. are not obviously better than the hydrolysis TaqMan only in the limited certain application field. The standard SYBR Green I real-time fluorescent PCR although solved the quantitative problem of endpoint PCR, but the basic protocol still didn't improved the non-specific amplification of the primer dimers after 30 cycle reaction of regular PCR. And hydrolysis TaqMan real-time PCR, which bypassed the primer dimers signal by adding target-specific probes hybridization, has one to two order magnitude better sensitivity than conventional PCR and loses something veracity of quantification due to its complexity and weak signal.
Other than the conventional endpoint PCR amplification only needs 25 to 30 cycles of product quantity enough, And real-time fluorescence PCR must detect low to 10 copies of near 40 cycles reaction. The TaqMan PCR requires at least 40 amplification cycles of reaction. The SYBR Green I real-time fluorescent PCR based on the DNA binding dye usually requires amplification 45 cycles for higher sensitivity and identify of non-specific PD. Therefore the real-time fluorescent PCR technology not only possesses the similar non-specification comparing with endpoint PCR but also the dye SYBR Green I real-time PCR couldn't distinguish the specific fragment with non-specific product at same time. All the non-specification also existed in any PCR system, but only the non-specification of exponential amplified PD which was from excess a pair of 3′ ends complement primers is a major source of non-specific product that comparable or catch up exponential amplification of target. A pair of excess primers due to the only 4 kinds of base combination has 25% and has 25% complementation on the other hand. So the primer pairs with the 3′ end of a small amount of base-pair complementary will cross hybridized and extend in the DNA polymerase PCR condition to form primer-dimer (PD), which used as next template is largely nonspecific amplified by free primers and bound to the DNA dye. The background Ct value (cycle threshold) of PD amplification of the vast majority primer pairs that carefully be optimization design, which 3′ end is less complement as possible, be located generally around 30 cycle in real-time PCR experiments without a template based dye SYBR Green I. Even some primers background Ct value is before 25 cycles that located in the Ct value 15-37 cycles of most target quantitative detection range or “gold detection window” and seriously covered the low concentration of molecular targets quantification. Although the primer dimer couldn't emit fluorescence in the TaqMan real time PCR, which the primer dimer problems is actually neglected, but it competitively inhibited 10 fold lower template amplification with same pairs of primer. Therefore sensitivity of TaqMan real time fluorescence PCR is only higher 10 fold one to order magnitude than the SYBR Green I real-time PCR. So far the very few reports of studies and resolve primer problem, current research still focus on the optimization design primer without the reverse complement and execute “hot-start” PCR for suppression of primer 3′ end of a few base-pair binding and extension in low temperature before amplification. On account of DNA polymerase immediately starts work when the PCR ingredients regularly put in pool at time. The so-called “hot-start” means the PCR reaction works to start amplification until components controllable releasing after the thermal denature in order to reduce non-specific reaction before PCR. The “hot-start” measures include using heat release of wax packaged magnesium Mg2+ ion, the modified suppression of Taq such as the N end missing of KlenTaq B., et al., 2003, Nucleic Acids Res., Vol. 31, No 21:6139-6147), the heat activated Taq with anti Taq antibody (Kellogg D E, al., 1994, Biotechniques 16:1134-1137) and Taq enzyme with inhibited Aptamar (Lin Y, et al., 1997, J. Mol Biol the heat activated primer with four oxidation pentane fixed (Lebedev A V, et al., 2008, Nucleic Acids Res., Vol. 36, No 20: and so on. The non-specific amplification especially PD come from the mispairing extension in the low temperature and as well in the higher annealing temperature of thermal-cycling reaction. The background Ct value of a artificial “hot-start” by manually adding a lacking component after denature to the PCR solution, which exactly lack the same component, is delayed 1-3 cycles comparing with un hot start common PCR and has about 32 cycles. And the efficiency of base-pair extension and activity of thermo Taq enzyme is usually low in the low temperature. The higher temperature of thermal cycling is absolutely major reason of PCR non-specific primer-dimer PD amplification. The Hands technique that is most close or similar with this current invention has adopt completely same sequence primers for eliminating of the non-specific PD amplification (Homo-Tag assisted non-dimer system, Brownie J., et al., 1997, Nucleic Acids Res., Vol. 25, No 16: p 3235-3241); And the chimeric DNA, RNA primer (Peleg, O., et al, Applied Enviro Micro-Bio., 2009, Vol. 75, No 19:6393-6398; and PCT: send 2009/004630); The oneself ends pairing of a single strand PD from completely same sequence primers can competitively bind free primers. And the chimeric primer with several RNA bases that cannot effectively be Taq polymerase template realizes the PD inhibition. But they not only inhibit the PD nonspecific, but also do not selectively interfere with the target specific amplification efficiency. The primer dimers amplified through primers 3′ base pairing as reciprocal primer and template. There is no intrinsic difference between target amplification by primer and by same primer pairing and amplification. The action is basically parallel, only the extent lighter. Any inhibition to the primer sequence would be no selectively inhibits target specific amplification.
The real time fluorescent PCR “closed tube analysis” in most cases is not real completely closed, there are also cross contamination of PCR products aerial fog pollution. In addition to the capillary tube with screw cap, most of the 0.2 ml PCR test tubes or 96 well plates will be soft and leak in the thermal cycling denature heating time. There will be some aerosol to leak out (squeeze) of the tube cap under the high temperature and high pressure. An aerosol particle could contain 105-106 amount molecules, which not only are the possible positive target amplicons and also contain high concentration of primer dimer amplification of excess primer pairs in every reaction well. As repeating the same PCR, the leaking pollution can be exponentially amplified again and be accumulated. Then the real-time fluorescent PCR is not start from 0 cycle but from the last end of past PCR cycles, the pollution like snowball grow more and more. To prevent aerosol recontamination of PCR products, the using of substrate dUTP instead of dTTP coupled with uracil-DNA-carbohydrate enzyme (UDG/UNG, U.S. Pat. No. 6,090,553), which is inactivated in later heating cycles, can selectively degrade the aerosol products of contamination in pre-cycling. But the UDG is double-sides sword to specific and nonspecific products, it less effectively degrade too much aerosol in less amount of UDG and over degrade target products in excess amount of UDG enzyme. The moderate amount of UDG which could eliminate small amount of cross contaminated aerosol in optimum temperature before PCR is not enough to stop the PD amplification and delay the background Ct value of SYBR Green I real-time fluorescent PCR in practical work.
In order to overcome the inherent obstacle of false positive of primer dimers PD of SYBR Green I real-time fluorescent PCR is not suitable to clinical testing analysis, as well as the limitation of the existing PD inhibition methods also similarly affect target specific amplification efficiency, this invention “Primer Middle Sequence Interference PCR Method” is on the basis of optimizing a pair of primers according to the conventional primer design principles. The not complement of intermediate domain (ID) of parallel primer pairs, or/and the interference of antisense base oligo-nucleotide complemented with the ID of primers, or/and the intra-primer interference of antisense sequence against ID of primer self, or/and above techniques combination, which maximum break the 3′end outside mis-binding join-force of primer 3′ends less-pairs need to borrowed in thermal cycling, then selectively inhibit the PD amplification without disturbing the target specific amplification efficiency. This invention of real time fluorescence PCR, which plus one end primer heat release and UDG pretreat-dUTP PCR system under the mineral-oil closing, will has not nonspecific primer dimer accumulation that background is a straight baseline in 45 PCR cycles. Multistep protected and closed PCR reaction does not produce the aerial fog or only the aerosol without effective amplification. Just in case the trace leak of PCR aerosol can further effective degrade by UDG enzyme. These integrated measures make sure the nucleic acid amplification test reliable without any false positive reaction.